Double-stranded cDNA fragments were synthesized from hepatitis A virus (HAV) RNA and inserted into the Pst I site of pBR322. The identity of cloned cDNA was established by demonstrating its hybridization to RNA from HAV-infected tissue culture cells but not to RNA from uninfected cells. Genomic length RNA of approximately 7500 nucleotides was the predominant species that hybridized with the cloned HAV cDNA. Cloned cDNA from near the 5' terminus of the genome was used to synthesize and clone cDNA by primer extension so that a molecular clone was obtained that contained the 5' terminus of the genome. Restriction endonuclease digestion and hybridization between subgenomic fragments yielded a map of overlapping cloned cDNAs of the complete viral genome. Cloned cDNA was used as a probe for detecting HAV RNA in tissue culture, serum, and fecal specimens by hybridization. Hybridization experiments also demonstrated that probes taken from any region of the HAV genome will not hybridize to RNA or cloned cDNA from a variety of other picornaviruses, a result that was supported by comparison of nucleotide sequences using computer programs. Ligation between overlapping cloned cDNAs resulted in a complete representation of the HAV genome in pBR322 and attempts are being made to produce virus from this clone by transfection.